玉米叶绿体铁氧还蛋白1的原核表达及部分性质分析

PROKARYOTIC EXPRESSION AND PARTIAL CHARACTERIZATION OF MAIZE CHLOROPLAST FERREDOXIN 1

叶绿体铁氧还蛋白(Fd)通过活性中心的铁硫簇传递还原力,在各种氧化还原途径中起重要作用。本研究中,氨基酸序列比对显示玉米中5种Fd的叶绿体导肽同源性很低,而去除导肽的成熟蛋白氨基酸序列具有很高的同源性。采用RT-PCR技术从玉米幼叶总RNA中克隆了编码成熟Fd1的基因,并分别插入pQE 80和p28SUMO表达载体,转化大肠杆菌BL21(DE3),表达的N端含有组氨酸标签的Fd1(His-Fd1),和含有组氨酸标签的小类泛素修饰蛋白(SUMO)的Fd1(HisSUMO-Fd1),用Ni-NTA层析介质亲和纯化,SDS-PAGE显示纯化的His-Fd1为一条带,亚基分子量约12kD。纯化的HisSUMO-Fd1用专一性蛋白酶Ulp除去HisSUMO,获得纯化的Fd1。紫外可见光谱扫描显示纯化的HisSUMO-Fd1溶液在315nm、415nm和459nm有特异吸收峰,电子顺磁共振波谱分析表明Fd1中存在[2Fe-2S]。SDS-PAGE显示多种玉米幼叶可溶性蛋白被固定化的His-Fd1吸附。

The ferredoxin（Fd） proteins in plant chloroplasts play important roles in cellular metabolism by delivering reducing equivalents through the [2Fe-2S]cluster in the active site to various essential oxido-reductive pathways.In this study,the chloroplast leading peptides from five Fd proteins of maize share the low homogeneity,whereas the mature Fd proteins deleted the leading peptides are high homogeneous,as displayed by the amino acid sequence alignments.The gene encoding mature maize ferredoxin 1（Fd1） was cloned by RT-PCR using the total RNA from young leaves as the template.The cloned gene was inserted into pQE80 and p28SUMO plasmid,and transformed into Escherchia coli BL21（DE3） respectively.The expressed Fd1 fused with the histidine-tag（His-Fd1） or HisSUMO tag（HisSUMO-Fd1） at N terminus,was purified by Ni-NTA affinity chromatography independently.The recombinant Fd1 protein was obtained by removing the HisSUMO using the specific protease Ulp.SDS-PAGE analysis showed that purified His-Fd1 has a molecular mass of about 12kD.The purified HisSUMO-Fd1 has the absorption peaks at 315,415 and 459 nm identified by UV-visible spectra scanning,and the [2Fe-2S]cluster determined by electron paramagnetic resonance（EPR） experiments.Several proteins from soluble extracts of young maize leaves were bound by the immobilized His-Fd1,as shown by SDS-PAGE analysis.